ALTERATION IN CHROMATIN CONFORMATION OF PROLIFERATING AND DIFFERENTIATING ENZYME GENES IN NORMAL MOUSE LIVER AND ASCITES HEPATOMA CELLS AND ITS RELATION TO THE GENE EXPRESSION OF ENZYMES

Nuclei from the normal mouse liver were partially digested with micrococcal nuclease, followed by DNA extraction, agarose gel clectrophoresis and dot blot hybridization with ~(32)P-labeled cDNA probes of CPS_1 and ACT complex, It was clearly shown that the CPS_1 genes were distributed on the monomer, dimer. and trimer of nucleosomes, while the genes coding for ACT complex were distributed on the condensed oligonucleosomes. An opposite manner of distribution of CPS_1 and ACT complex genes was, however, noted in the case of ascites hepatoma cells, in which the specific activity of ACT was 13 times higher than that in the normal liver, while that of CPS_1 was remarkably reduced. Similar patterns of change in mRNA level of CPS_1 and ACT complex were observed in the normal mouse liver and ascites hepatoma cells, indicating a close relationship between chromatin structure and gene expression of these enzymes.     

STUDIES ON THE MECHANISM OF CARCINOGENESIS(IX)——DIFFERENTIAL EXPRESSION OF PROLIFERATING AND TISSUE-SPECIFIC ENZYME GENES-DURING HEPATOCARCINOGENESIS

With cDNA fragments of CPSI, OCT and ACT as probes, dot and Northern blot analyses of poly(A)+-RNA from rat liver with different pathological lesions during carcinogenesis induced by di-ethylnitrosamine were conducted. It was shown that the level of mRNA of tissue-specific enzymes, CPSI and OCT decreased while that of the proliferating enzyme ACT mRNA increased, and the alteration was correlated with the degree of pathological changes in each case. The relative changes in the mRNA level of these enzymes during hepatocarcinogenesis were correlated with that of enzyme activities. Implication of these findings in the mechanism of carcinogenesis in terms of cell proliferation and differentiation was discussed.     

小鼠腹水肝癌细胞增殖酶及分化酶基因染色质构象的变化及其与酶基因表达的关系

小鼠腹水肝癌细胞的CPS_1活性较正常肝显著降低,而ACT活性则增高,约为正常肝的13倍。RNA-cDNA斑点杂交证明,肝癌中CPS_1mRNA很低,而ACTmRNA较正常肝增多。以微球菌核酸酶限制性消化正常肝及肝癌细胞核,琼脂糖凝胶电泳以及DNA-cDNA斑点杂交结果表明,在正常肝中,表达活跃的CPS_1基因分布在结构松散的单体、二聚体、三聚体核小体上,而表达较低的ACT基因则分布在比较致密的寡聚核小体上。腹水肝癌细胞的情况正好相反。CPS_1基因的表达很低,主要分布于寡聚核小体,而ACT基因的表达增高,主要分布于单体核小体。这些结果充分说明,肝癌细胞中分化酶CPS_1基因与增殖酶AST基因表达的相互变化与这两种基因核小体构象的相互变化有密切关系。     

PURIFICATION AND CHARACTERIZATION OF A DNA POLYMERASE-TEMPLATE COMPLEX IN MOUSE ASCITES TUMOR CELLS

A DNA polymerase-template complex found in mouse Erhlich ascites tumor cells waspurified by twice discontinuous gradient centrifugations and agarose gel filtration. Afterpurification, the specific activity of the complex increased about 500-fold. The size of thecomplex was found to be 510,000 daltons. The shape of the complex was globular under electronmieroscope. Its diameter was between 100--110 A. The complex was isolated into two mainenzyme proteins and a homogeneous DNA template by DEAE cellulose chromatography. Themain enzyme had a molecular weight of 300,000 daltons and a sedimentation coefficient of9.5s. The endogenous template DNA showed a single zone in polyacrylamide gel electropho-resis analysis. The sedimentation coefficient was determined to be 3.8s.     

CLONING OF cDNA CODING FOR CARBAMYL PHOSPHATE SYNTHETASE I AND CHANGES IN LEVELS OF CPS_1 mRNA DURING HEPATOCARCINOGENESIS

cDNA coding for carbamyl phosphate synthetase I was cloned from recombinant plasmid with insertcomplementary to the mRNA for CPS_1 followed by hybrid-selected translation screening. The length ofthe insert CPS_1 cDNA was approximately 800 base pairs. Using this cDNA as a probe, it was foundby dot-blot analysis of the total RNAs and poly(A)~+-RNAs isolated from rat livers with different path-ological lesions induced by diethylnitrosamine that the levels of CPS_1 mRNA were decreased, the de-crease being correlated with the malignancy of hepatocytes during carcinogenesis.     

CPS_1 cDNA克隆及大鼠肝癌癌变过程中CPS_1 mRNA量的变化

本研究以CPS_1mRNA为模板合成cDNA,与质粒pBR 322 DNA重组,经选择杂交法筛选获得GPS_1 cDNA克隆。以~(32)P标记cDNA作为探针与二乙基亚硝胺(DENA)诱发大鼠肝癌过程中不同病变肝中提取的总RNA及poly(A)~+RNA杂交。结果发现,CPS_1 mRNA的量随肝细胞癌变程度加深而递减,说明肝癌癌变过程中,CPS_1 基因表达的异常可能发生在转录水平。     

小鼠腹水癌细胞中一种DNA聚合酶-模板复合体的纯化和特性鉴定

本文通过两次不连续密度梯度离心和琼脂糖凝胶过滤,对小鼠腹水癌细胞中的DNA聚合酶一内源模板复合体进行了纯化,纯化后酶活性提高了500倍,经鉴定其分子大小为510,000道尔顿。电子显微镜下观察呈均匀球状,直径100—110埃。复合体经DEAE纤维素柱层析分离为两种酶蛋白组份和一种DNA模板。其中最主要的酶蛋白分子量为300,000道尔顿,沉降系数为9.5s;内源DNA模板经聚丙烯酰胺凝胶电泳分析呈单一区带,沉降系数为3.8s。     

癌变原理研究——Ⅸ.大鼠肝癌癌变过程中细胞增殖酶和组织特异酶基因表达的相互变化

以CPS_1,OCT,ACT cDNA克隆片段为探针与二乙基亚硝胺诱发大鼠肝癌癌变过程不同病变肝组织的poly(A)~+-RNA进行斑点杂交及Northern印迹杂交。结果表明:组织特异酶CPS_1,OCT mRNA量降低,而细胞增殖酶ACT mRNA量升高。这些mRNA量的变化都随病变程度而加深。CPS_1 mRNA分子略大于28S,OCT mRNA约为15S,ACT mRNA分子约为35S。癌变过程中CPS_1,OCT以及ACT mRNA量的相互变化与不同癌变肝组织酶活性的相互变化呈相关关系。